Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope. However, the FlashLab technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance - a cluster is formed, and the presence of chromogenic proteins allows the user to spot it in the naked eye, without the need for a complex device (for more information about our chip click here).
Three chromogenic proteins (chromoproteins) were tested for the S.Tar
system, all which were provided and extracted from the iGEM 2016 kit.
Each part contained RBS, chromoproteins coding sequence and a double
terminator. The different parts contained the following proteins:
- tsPurple, visible as purple color (K1357008).
- amilCP, visible as blue color (K1357009).
- mRFP, visible as red color and can serve, also ,as red fluorescence protein (K1357010).
To test the expression and visibility of those proteins, a strong promoter (J23100) was cloned upstream to the RBS using the RFC10 assembly (Fig. 1).
This plasmid is one of two plasmids constructing our FlashLab system, as
the other is plasmid expressing a chemoreceptor. The two plasmids were
co-transformed to UU1250 strain expressing both, the designed
receptor and a chosen color.
Each plasmid contains different antibiotic resistance allowing easy screening for strain expressing both proteins.
The first step, as mentioned in the implementation section, was to clone a strong promoter (J23100) upstream to each part, creating a high expression system. The biological system was then transformed to E.coli Top10 strain and UU1250 strain. Plating results showed colored colonies, for both strains, as expected. Colored colony from each type was incubated overnight at 37℃ in LB medium. Overnight incubation resulted in a medium that appeared as colored, due to high concentration of bacteria expressing chromoproteins. The results of centrifuging the medium sample was a colored pellet (Fig. 1).
As both strains showed similar results, the following experiments conducted
only with the UU1250 strain, the strain which was used for chemotaxis assays.
Growth conditions for chemotaxis assays require a minimal growth medium,
TB, and a temperature of 30℃. Above this temperature the bacteria lose their chemotaxis ability.(1).
Overnight growing in this condition, of UU1250 strain expressing chromoproteins resulted in a colorless medium, although bacterial concentration was high. In order to overcome this issue, two different growth conditions were tested. Incubation at 37℃ in TB medium and incubation at 30℃ in LB medium. At 37℃ TB medium, color was detected. The color was less intense compared to the 37℃ LB medium, but still high enough to be detected by a naked eye. Moreover, the pellet's color intensity was similar to the one grown 37℃ LB pellet. As for the 30℃ LB medium, no color was detected after overnight growth. In addition, the pellet was also colorless.
These results imply that the growth temperature has a significant influence on the chromoprotein expression. To achieve color intensity at the right conditions a two-stage growth was conducted. The first stage is incubation at 37℃ in LB medium in order to gain a high expression of chromoproteins. The culture is then centrifuged and resuspend with TB medium. The second stage is incubation at 30℃ for 3 hours, for restoring chemotaxis abilities. This two-stage growth allows both color expression and chemotaxis ability to the bacteria, and was proven to be effective in matter for chromoprotein expression, as for chemotaxis ability test, the two palsmid system was conducted.
The two plasmid system
FlashLab system is based on a two-plasmid system, where motile bacteria express both chromoproteins, using two different expression plasmids. In order to verify the idea, Tar was chosen as the chemoreceptor and amilCP (blue color) as the chromoprotein. The first plasmid (K1992004) expresses the Tar chemoreceptor, along with chloramphenicol (CM) resistance. The second plasmid expresses a chromoprotein (see Implementation – part have not been submitted) along with ampicillin (Amp) resistance. Follolwing transformation of the two plasmids into UU1250 strain (which lacks chemoreceptors genes), the colonies were selected by plating on LB agar plates with two antibiotic. The plating results showed colored colonies and non-colored ones (Fig. 2), that is due to the non-compatible ORI of the two plasmids (see Outlook section).
Fig. 2: Co-transformation of K1992004 and tsPurple expressing plasmid to UU1250 strain.
The colored colonies were isolated and grown, using the two-stage growth method mentioned previously. The result is high density and colored medium (Fig. 3).
Fig. 3: Left tube - UU1250 strain expressing Tar chemoreceptor only (K1992004). Right tube - UU1250 strain expressing Tar chemoreceptor and tsPurple chromoprotein.
The sample was then tested for chemotaxis ability using swarming assay, in which the chemotaxis ability is examined in a swarm plate. The bacteria depleting the attractant and move outwards, creating a halo. (Fig. 4). A halo was formed after 8 hours indicating functional chemotaxis response. For our chip assay more intense color was needed. Thus, the sample was centrifuged and resuspended in a smaller volume of TB medium, increasing the bacterial concentration by 10-folds. Results can be seen in the Proof of concept page.
We succeeded to get colored bacteria, grown in the optimal condition of our assay, though both the chemoproteins and the receptors were cloned on high copy plasmid with the same ORI - pMB1 (pSB1C3 and pSB1A2). Usually, plasmids with the same ORIs are incompatible, because they will compete for the same machinery, creating an unstable and unpredictable environment. For future plan, we suggest to clone one of the expression systems to a plasmid containing different ORI, compatible to pMB1 ORI. This adjustment will improve the stability of our system, and allow better control over the expression of each protein.
1. MAEDA, Kayo, et al. Effect of temperature on motility and chemotaxis of Escherichia coli. Journal of bacteriology, 1976, 127.3: 1039-1046.